Abstract: Contrary to long-standing assumptions, bacteria communicate extensively to coordinate sophisticated multi- cellular behaviors such as antibiotic production, toxin secretion, motility, biofilm formation, symbiosis, and de- velopment. The best understood examples of bacterial communication occur at a distance via diffusible signaling molecules. Recent evidence indicates that bacteria can also be more intimately connected, in some cases directly exchanging cytosolic contents; however, the extent, consequence, and molecular underpinnings of these intimate modes of bacterial cell-cell communication remain poorly understood. Here I propose an in- novative and multi-disciplinary line of investigation to test a paradigm-shifting feeding tube model for bacterial cell-cell communication in which direct intercellular metabolite exchange drives developmental gene expres- sion during spore formation by the model bacterium Bacillus subtilis. More specifically, the central hypothesis of this proposal is that the developing spore (the forespore) is programmed to lose its metabolic self-sufficiency at intermediate stages of sporulation and that a novel channel apparatus acts as a portal (a feeding tube) through which the adjacent mother cell replenishes the small molecule resources generally required for late forespore developmental gene expression. To test this hypothesis, we will first comprehensively characterize the metabolic status of the forespore in the presence and absence of the channel using novel in vivo biosen- sors and global metabolite profiling. A reverse genetic screen will then be undertaken to identify the genetic circuit responsible for forespore metabolic shutdown. Second, we will develop and implement novel cytological assays to demonstrate the ability of small molecules to transit from the mother cell to the forespore in a feeding tube dependent manner. Finally, we will determine the structure of the feeding tube channel and key protein components thereof by cryo-electron microscopy and X-ray crystallography. In all, the proposed research is expected to have a profound impact on our understanding of this remarkable example of bacterial communica- tion as well as similarly intimate modes of bacterial cell-cell communication that have only recently come to light. This knowledge will be significant because it will address fundamental questions regarding bacterial inter- connectedness in nature, including during horizontal gene transfer, pathogenesis, and biofilm formation. This work also has high potential to identify novel antimicrobial targets in a broad range of bacteria and especially gram-positive, spore-forming pathogens related to B. subtilis such as B. anthracis, Clostridium botulinum, and C. difficile. Finally, this research plan is innovative not only because it will elucidate the molecular underpin nings of a remarkably intimate and unexpected mode of bacterial cell-cell communication and developmental gene regulation, but also because it will implement a unique combination of traditional and cutting-edge tech- nologies to accomplish this goal. Public Health Relevance: Recent evidence indicates that bacteria can be highly interconnected in nature, in some cases directly exchanging cytosolic contents; however, the extent, consequence, and molecular underpinnings of these intimate modes of bacterial cell-cell communication, including during horizontal gene transfer, pathogenesis, and biofilm formation, remain poorly understood. Here I propose an innovative and multi-disciplinary line of investigation to characterize a remarkable mode of bacterial cell-cell communication in which direct intercellular metabolite exchange drives developmental gene expression in the model bacterium Bacillus subtilis. This study will transform our understanding of intimate bacterial connections and has high potential to identify novel antimicrobial targets in a broad range of bacteria including gram-positive, spore-forming pathogens related to B. subtilis such as B. anthracis and Clostridium difficile.